It is well known that certain bacteria of the genus Xanthomonas, notably the Xanthomonas campestris group, when cultured under suitable conditions are capable of producing hetero-polysaccharides, commonly termed "Xanthan gum", which are useful as thickeners and emulsifiers in a variety of applications including foodstuffs and drilling muds. In general the bacteria are grown in batch culture, typically for 48 to 72 hours, in conventional complex culture media containing nutrients such as corn starch (U.S. Pat. No. 3,455,786), soy peptone (U.S. Pat. Nos. 3,391,060 and 3,391,061) distillers solubles (French Pat. No. 2,251,620) or "Stimuflav" (U.S. Pat. No. 3,020,206).
U.S. Pat. No. 3,391,060 (McNeely) described the use of ammonium nitrate as a nitrogen source in the final culture medium, but observes that to build up the bacterial population to a satisfactory level a complex nitrogen source is essential in the seed cultures and may also be used in the final culture in addition to ammonium nitrate. Thus U.S. Pat. No. 3,391,060 employs a semi-, rather than a fully, defined culture medium containing the ammonium nitrate of the final fermentation medium and the complex nitrogen sources of the seed cultures. That this was the intention of the patentee is illustrated by a later publication (Industrial Gums, 2nd Edn, Academic Press, 1973 at p 488) in which McNeely states that an inorganic nitrogen source and a protein supplement are necessary for efficient polysaccharide production.
Similarly, Starr (J Bact., 1946, 51, 131), whilst reporting that certain strains of Xanthomonas will grow in a defined medium, concludes that the rate of bacterial growth is considerably faster in a complex medium. Furthermore, the authoritative Bergey's Manual of Determinative Bacteriology (8th Edn) states that the minimal growth requirements of Xanthomonas are complex. It should also be noted that Starr does not address the question of polysaccharide production. It is not enough that a Xanthomonas strain will grow in a particular medium, it must also efficiently produce polysaccharide in that medium.
Many applications of xanthan gum require large quantities of the material and therefore considerable attention has been given to the economics of its production.
Batch production of xanthan gum requires at least 48 to 72 hours to achieve maximum yield, whilst similar yields per unit volume of culture may be obtained in 12 to 50 hours by continuous methods. The desirability of producing xanthan gum by continuous method was therefore recognised at an early stage (U.S. Pat. No. 3,328,262, Lindblom and Patton). However successive attempts to produce an economically viable system (U.S. Pat. No. 3,485,719, Rogovin; Silman and Rogovin, Biotechnol. Bioeng, 1970, 12, 75 and 1972, 14, 23) were unsuccessful because the bacteria: (Xanthomonas), under the conditions employed, developed into strains that did not produce polysaccharide. (It should be noted that to compete with a batch system, a continuous culture should proceed for at least 10 turnovers of culture medium (culture turnover=Q=length of culture.times.dilution rate), and preferably for much longer than that).
It had long been known that only certain strains of Xanthomonas bacteria were capable of producing polysaccharide, the suitable strains normally being those which produce smooth globular colonies when plated onto nutrient agar containing glucose. Attempts to produce polysaccharide by the continuous culture of these "smooth strains" showed that, although cell growth proceeded as expected, polysaccharide yield fell to unacceptably low levels after a short time; this fall was attributed to the development, as the culture progressed, of non-polysaccharide producing Xanthomonas strains.
Thus Silman and Rogovin (supra) concluded that cultures could not be continued beyond 6.5 to 8.7 culture turnovers, whilst Rogovin (supra) noted that in his process (which was not truly continuous); "contamination" occurred after 6.5 culture turnovers.
Thus despite numerous attempts (see also U.S. Pat. No. 3,328,262 and U.S. Pat. No. 3,281,329 (Lipps)), no successful process for producing polysaccharides by a simple, single stage continuous culture of Xanthomonas bacteria has so far been described.
The present inventors have now found that by employing a different culture medium from that used by Silman, Rogovin, etc, Xanthomonas strain variability in continuous culture may be substantially reduced and fall-off in polysaccharide yield thereby substantially avoided.
The medium chosen by the present inventors is a chemically defined culture medium. This replaces the complex media favoured in the prior art processes. Surprisingly, and contrary to the teaching of Bergey and of McNeely the use of a chemically defined medium, as taught herein, adversely affects neither bacterial cell growth nor polysaccharide production.